WO2022059498A1 - Dispositif de formation de tissu musculaire tridimensionnel et procédé de production de tissu musculaire tridimensionnel - Google Patents

Dispositif de formation de tissu musculaire tridimensionnel et procédé de production de tissu musculaire tridimensionnel Download PDF

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WO2022059498A1
WO2022059498A1 PCT/JP2021/032244 JP2021032244W WO2022059498A1 WO 2022059498 A1 WO2022059498 A1 WO 2022059498A1 JP 2021032244 W JP2021032244 W JP 2021032244W WO 2022059498 A1 WO2022059498 A1 WO 2022059498A1
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muscle tissue
culture
support member
culture solution
connector
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PCT/JP2021/032244
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English (en)
Japanese (ja)
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昌治 竹内
雄矢 森本
亜衣 島
康彬 石井
優介 平田
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日清食品ホールディングス株式会社
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Priority to US18/245,455 priority Critical patent/US20240026260A1/en
Priority to JP2022550456A priority patent/JPWO2022059498A1/ja
Publication of WO2022059498A1 publication Critical patent/WO2022059498A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/40—Manifolds; Distribution pieces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/10—Perfusion
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present invention mainly relates to a three-dimensional muscle tissue construction device and a method for manufacturing a three-dimensional muscle tissue.
  • Meat demand is increasing with the increase in population and income in emerging countries.
  • it is difficult to increase the supply of meat due to the soaring price of grains used as feed for livestock and the problem of securing a breeding place, and the development of alternative meat is expected.
  • Cultured meat is made by forming tissue using skeletal muscle cells increased by culture. Since it can be produced in the laboratory, it can be produced without being affected by climate change, and has less greenhouse gas emissions and less environmental load than conventional livestock.
  • An object of the present invention is to provide a three-dimensional muscle tissue constructing device for manufacturing a three-dimensional muscle tissue and a method for manufacturing a three-dimensional muscle tissue.
  • the present invention provides the following three-dimensional muscle tissue constructing device and a method for manufacturing a three-dimensional muscle tissue.
  • a first muscle tissue anchor engaged with a first support member and / or a first connector A three-dimensional muscle tissue building device with a second muscle tissue anchor engaged to a second support member and / or a second connector.
  • the three-dimensional muscle tissue construction device according to [1], further comprising a first support member and a flow path forming member for a culture solution that engages with the second support member.
  • the three-dimensional muscle tissue construction device according to [1] or [2], further comprising a pump for supplying the culture solution to the culture solution inlet.
  • a hydrogel containing skeletal myoblasts is obtained with the flow path forming member inserted through the first support member and the second support member.
  • a method for producing three-dimensional muscle tissue which comprises a step of supplying to and culturing skeletal myoblasts in three dimensions.
  • the present invention specifically provides a three-dimensional muscle tissue construction device and a manufacturing method.
  • a three-dimensional muscle tissue having a sufficient thickness can be produced. Therefore, the three-dimensional muscle tissue produced by the present invention can be expected to have a texture similar to that of edible steak meat. Further, in regenerative medicine applications, by using thick three-dimensional muscle tissue, it can be expected that damage to a wide range of thick muscle tissue can be repaired with a single treatment.
  • the schematic diagram which shows the outline of the 3D muscle tissue construction device of this invention.
  • An embodiment of the three-dimensional muscle tissue construction device of the present invention is shown.
  • Procedure for forming a three-dimensional muscle tissue-building device during parallel culture Procedure for forming a three-dimensional muscle tissue-building device during vertical culture.
  • V and H represent the vertical direction and the horizontal direction, respectively.
  • HE-stained image Comparison of (a) perfused tissue, (b) non-perfused tissue, and (c) cell concentration.
  • Comparison of (a) perfused tissue, (b) non-perfused tissue, and (c) cell concentration Photographs of the tissue surface and interior. Orientation index inside the tissue.
  • the three-dimensional muscle tissue construction device 1 of the present invention includes a first connector 4 and a second connector 5 facing each other, and a flow path 3 of a culture solution is formed between the first support member 8 and the second support member 9. There is.
  • the flow path 3 has a solid flow path forming member 10 (FIG. 5B) such as a wire, a wire, and an acupuncture beam between the first support member 8 and the second support member 9. It can be formed by filling the culture space 2 between the first connector and the second connector with a hydrogel containing skeletal myoblasts in the inserted state, and pulling out the flow path forming member 10 after the hydrogel has hardened. ..
  • the flow path 3 in this case is a cavity between the first connector 4 and the second connector 5 formed in the three-dimensional muscle tissue 16.
  • the channel forming member 10 Since the channel forming member 10 is withdrawn after injecting a hydrogel containing skeletal myoblasts into the culture space, it is preferable to coat the surface thereof with a material that suppresses cell adhesion, for example, bovine serum albumin (BSE).
  • BSE bovine serum albumin
  • Hydrogels containing skeletal myoblasts have fluidity that allows injection when injected into the culture space, but after a while the gel hardens to gain sufficient strength and shape retention.
  • the hole through which the flow path forming member 10 of the first connector 4 passes is closed with the sealing material 20.
  • the second connector 5 has a hole for passing the flow path forming member 10
  • the hole for passing the flow path forming member 10 of the second connector 5 may be similarly closed with the sealing material 20.
  • the first connector 4 and the sealing material 20 may be integrally molded. Even if the first connector 4 and the sealing material 20 are integrated, there is no particular problem as long as the culture solution can be circulated.
  • the flow path 3 has a gas component such as oxygen or carbon dioxide and / or a nutrient 14 (hereinafter, “nutrient, etc.” between the first support member 8 and the second support member 9).
  • a hollow flow path forming member 10 through which waste products can pass is attached, and the culture solution 15 is allowed to flow inside the flow path forming member 10, so that nutrients and the like can be contained inside the three-dimensional muscle tissue. 14 can be supplied and carbon dioxide and waste products can be discharged.
  • the flow path 3 of the culture solution 15 in this case is a cavity inside the flow path forming member 10.
  • the culture solution 15 is supplied to the culture solution introduction port 6 by the pump 12, flows from the first support member 8 into the second support member 9 through the flow path 3, and contains waste products that have passed through the second support member 9.
  • the culture solution 15 is discharged from the culture solution discharge port 7 formed in the second connector 5.
  • the first support member 8 and the second support member 9 are provided with an opening for inserting the flow path forming member 10.
  • the encapsulant 20 is a encapsulant for encapsulating the opening on the first support member side, and the encapsulant 20 allows the culture broth 15 introduced from the culture broth introduction port 6 to pass through the flow path 3. It flows in the direction of the arrow in FIG.
  • the sealing material 20 for example, Ecoflex (registered trademark) manufactured by BASF can be used.
  • the opening 5a on the second support member side is not sealed in FIGS. 1 and 2, a part of the culture solution that has passed through the second support member 9 is directly discharged from the culture solution discharge port 7. A part of the mixture is mixed with the culture solution 15 outside the culture space and discharged from the culture solution discharge port 7.
  • the opening on the side of the second support member 8 may be sealed with the sealing material 20.
  • the culture solution may be discharged from the culture solution discharge port 7, but by arranging the tip of the discharge tube in the culture solution outside the muscle tissue 16, the culture solution outside the muscle tissue and the flow path 3
  • the culture broth containing the waste products that have passed through the above may be mixed, and the mixed culture broth may be discharged.
  • the surface of the culture (three-dimensional muscle tissue 16) of the skeletal myoblast 13 is sufficiently supplied with nutrients 14 from the culture solution 15, and the inside of the muscle tissue 16 is nutrients from the fresh culture solution flowing through the flow path 3.
  • Etc. 14 are supplied, and the waste liquid having a high concentration of carbon dioxide and waste products is discharged from the culture liquid discharge port 7.
  • the culture broth circulates, but the culture broth discharged from the culture broth outlet may be discarded and fresh culture broth may be supplied from the culture broth introduction port 6 by the pump 12.
  • a syringe pump can be used as the pump 12.
  • the pump 12, the culture solution introduction port 6, and the culture solution discharge port 7 may be connected by a tube.
  • the system of the present invention may include electrodes (not shown).
  • the construction of muscle tissue can be promoted. If the culture solution 15 is sufficiently abundant in the culture tank, the culture solution 15 may be circulated, but if the culture solution 15 is small, the old culture solution 15 that has passed through the flow path 3 should be discarded. Is desirable.
  • first support member and the second support member are arranged on a straight line to form a straight flow path.
  • the distance between adjacent flow paths depends on the thickness (diameter or cross-sectional area) of the flow paths, but is preferably about 500 to 1500 ⁇ m.
  • the peripheral portion of the muscle tissue 16 is sufficiently supplied with nutrients 14 from the culture solution 15, so that the skeletal myoblast 13 proliferates well. Since the supply of nutrients and the like 14 is insufficient inside the muscle tissue 16, the thicker the muscle tissue 16 is, the more likely the cultured cells are to die. In the present invention, nutrients and the like 14 are evenly supplied to the periphery and the inside of the muscle tissue 16, and carbon dioxide and waste products are discharged. Therefore, a thick muscle tissue 16 can be obtained in a short period of time, and the surface and the inside muscle tissue 16 can be obtained. The difference does not matter.
  • the shapes of the first and second muscle tissue anchors 11 are not particularly limited, but mesh-like ones as shown in FIGS. 4A to 4C are preferable.
  • the muscle tissue 16 is entwined with the mesh-shaped anchor 11 and both ends are fixed.
  • the skeletal myoblast 13 contracts when the culture is continued, but is fixed by the anchors 11 on both sides, so that the thick muscle tissue 16 can be maintained even if the culture is continued. Since the skeletal myoblast 13 contracts as the culture is continued, in FIGS. 1 to 3, the vicinity of the central portion is slightly recessed.
  • electrical stimulation may be applied to promote the proliferation of the skeletal myoblast 13.
  • FIG. 5 and 6 schematically show the procedure of the method for producing a three-dimensional muscle tissue of the present invention.
  • the first connector 4, the second connector 5, and the muscle tissue anchor 11 are fixed to the base material 17.
  • the culture space 2 is formed by inserting the flow path forming member 10 through the first support member 8 and the second support member 9 and further fixing the two front and rear side walls 18 to the base material 17.
  • FIG. 5C is a diagram when the culture space 2 is filled with a hydrogel containing skeletal myoblast 13.
  • FIG. 6A shows a state in which the flow path forming member 10 is pulled out and the two front and rear side walls 18 are further removed.
  • FIG. 6B the culture solution is flowed from the culture solution introduction port 6 toward the culture solution discharge port 7, and the culture is started.
  • Hydrogel containing skeletal myoblast 13 was injected into the gap of the culture space 2 in which the muscle tissue anchor 11, the first support member 8, the second support member 9, the flow path forming member 10, and the like were housed. After standing for a while, the gel solidifies and retains its shape, after which the side wall 18 is removed.
  • the time required for the gel to harden varies depending on the type of hydrogel and the environment, and therefore needs to be adjusted as appropriate. For example, about 30 minutes for collagen gel and about 12 hours for matrigel.
  • FIG. 6 is a diagram when the culture solution is flowed in the horizontal direction
  • FIG. 7 is a diagram when the culture solution is flowed in the vertical direction.
  • the embodiment shown in FIG. 7 is preferable because there is no possibility that the flow path is narrowed by the action of gravity of the muscle tissue.
  • the oxygen concentration in the culture solution is preferably 80% or more of the saturated oxygen concentration.
  • the muscle tissue anchor can be coated with a biocompatible material to enhance the adhesion of skeletal myoblasts and prevent the muscle tissue from falling off during culture.
  • biocompatible materials include fibronectin.
  • myotubes are formed when the culture of skeletal myoblasts is continued.
  • Hydrogels used for culturing skeletal myoblasts include fibrin, fibronectin, laminin, collagen (eg, type I, type II, type III, type V, type XI, etc.), agar, agarose, glycosaminoglycan, etc. Hydrogels of components constituting the extracellular basement membrane matrix such as hyaluronic acid and proteoglycan can be used. Commercially available products can also be used as hydrogels, for example, components based on mouse EHS tumor extracts (including type IV collagen, laminin, heparan sulfate proteoglycan, etc.) sold under the trade name "Matrigel". Can be done.
  • collagen includes undenatured collagen and denatured collagen.
  • Gelatin is exemplified as the denatured collagen.
  • the hydrogel preferably contains collagen, preferably undenatured type I collagen, especially when the skeletal myoblasts are derived from bovine.
  • type I collagen is contained, the content thereof is preferably 0.3 mg / mL or more, more preferably 1.0 to 3.0 mg / mL, and further preferably 1.0 to 1.5 mg / mL.
  • the skeletal myoblasts in the hydrogel have, for example, a cell concentration of about 1.0 ⁇ 10 6 cells / ml or more, preferably about 1.0 ⁇ 10 7 cells / mL to about 1.0 ⁇ 10 8 cells / mL, more preferably 5.0 ⁇ 10 7 It is preferably cells / mL to about 1.0 ⁇ 10 8 cells / mL.
  • Skeletal myoblasts contained in hydrogel can be prepared by a known method.
  • primary myoblasts obtained by treating a muscle tissue derived from a living body with a degrading enzyme for example, collagenase
  • Primary myoblasts are preferably filtered to remove impurities such as connective tissue.
  • skeletal myoblast cells derived from somatic stem cells capable of differentiating into pluripotent stem cells such as ES cells and iPS cells and skeletal myoblasts can also be used.
  • Skeletal myoblasts are derived from vertebrates such as mammals, birds, reptiles, amphibians, and fish.
  • mammals include non-human mammals such as monkeys, cows, horses, pigs, sheep, goats, dogs, cats, guinea pigs, rats, and mice.
  • birds include ostriches, chickens, ducks, sparrows and the like.
  • reptiles include snakes, crocodiles, lizards, turtles and the like.
  • amphibians include frogs, newts, salamanders and the like.
  • fish include salmon, tuna, shark, Thailand, and carp.
  • the skeletal myoblasts are preferably derived from mammals bred for livestock such as cattle, pigs, sheep, goats, and horses, and more preferably derived from cattle.
  • a skeletal myoblast genetically modified by a genome editing method such as a homologous recombination method or a CRISPR / Cas9 method or a non-genetically modified skeletal myoblast can be used.
  • a genome editing method such as a homologous recombination method or a CRISPR / Cas9 method
  • a non-genetically modified skeletal myoblast can be used.
  • the culture medium can contain medium components (for example, various amino acids, inorganic salts, vitamins, etc.), serum components (for example, growth factors such as IGF-1, bFGF, insulin, testosterone, etc.), antibiotics, and the like.
  • medium components for example, various amino acids, inorganic salts, vitamins, etc.
  • serum components for example, growth factors such as IGF-1, bFGF, insulin, testosterone, etc.
  • antibiotics and the like.
  • the three-dimensional muscle tissue mainly means artificially manufactured muscle that is not derived from a living body.
  • the three-dimensional muscle tissue of the present invention is composed of skeletal muscle cells (striated muscle cells).
  • Skeletal muscle cells are a form of myotubes (myotube cells) or muscle fibers in which myoblasts, which are precursors thereof, are multinucleated.
  • myofibrils are composed of myofibrils composed of actin fibers (actin filaments), which are proteins constituting muscles, and myosin fibers (myosin filaments), which are proteins constituting muscles. Furthermore, myofibrils have a structure in which a plurality of sarcomere structures are connected in the long axis direction. It is known that muscle contraction and relaxation occur based on the interaction (slip) of actin and myosin in sarcomere.
  • the preferred three-dimensional muscle tissue of the present invention has a sarcomere structure. However, it does not matter whether or not slippage occurs in the sarcomere structure.
  • Whether or not the three-dimensional muscle tissue has a sarcomere structure can be evaluated by a known method. For example, the presence of sarcomeric ⁇ -actinin (SAA), which is a protein constituting the Z membrane of sarcomere structure, was evaluated by immunostaining for SAA, and SAA immunostaining was positive and SAA was distributed in regular stripes. If so, it can be determined to have a sarcomere structure.
  • SAA sarcomeric ⁇ -actinin
  • the muscle fibers are aligned and oriented in the same direction.
  • the orientation of muscle fibers can be assessed, for example, by immunostaining with SAA.
  • the components (preferably all components) used in the production method of the present invention satisfy predetermined criteria and are used for food production and / or food. It is preferable, but not limited to, the ingredient in which the safety of the above is ensured.
  • Skeletal myoblasts can be cultured, for example, in the above-mentioned medium for growth culture by a method known to those skilled in the art.
  • a suitable culturing method a method of culturing under conditions of about 37 ° C. and a carbon dioxide concentration of about 5 to 10% (v / v) is exemplified, but the method is not limited thereto. Culturing under the above conditions can be performed using, for example, a known CO 2 incubator.
  • serum components for example, horse serum (Horse)
  • a normal liquid medium such as DMEM (Dulbecco's Modified Eagle's Medium), EMEM (Eagle's minimal essential medium), ⁇ MEM (alpha Modified Minimum Essential Medium).
  • Serum Fetal bovine serum (FBS), human serum (Human Serum), etc.), components such as growth factors; culture medium supplemented with antibiotics such as penicillin and streptomycin can be used.
  • fetal bovine serum When adding a serum component to a medium for growth culture, fetal bovine serum can be used as the serum component.
  • concentration of serum components can be about 10% (v / v).
  • the culture period can be, for example, about 1 day to 2 weeks.
  • skeletal myoblasts can be induced to differentiate into myotubes.
  • This differentiation induction skeletal myoblasts are multinucleated by cell fusion with surrounding cells, and myotubes are formed.
  • the myotubes form muscle fibers as they mature further.
  • the above culture can be carried out, for example, in a medium for inducing differentiation (culture for multinucleation) by a method known to those skilled in the art.
  • a suitable culturing method a method of culturing under conditions of about 37 ° C. and a carbon dioxide concentration of about 5 to 10% (v / v) is exemplified, but the method is not limited thereto. Culturing under the above conditions can be performed using, for example, a known CO 2 incubator.
  • myoblasts become low in nutrients, they involve surrounding cells and start polynuclearization. Therefore, it is preferable to induce differentiation into myotubes using a medium having less nutrients than the above-mentioned growth culture. Since horse serum is known to have less nutrients than fetal bovine serum, horse serum can be used. The concentration of serum components can be about 2% (v / v).
  • the flow path through which the culture solution flows may be in the horizontal direction or in the vertical direction. It is preferable that the flow path is in the vertical direction because the flow path can be prevented from being narrowed by its own weight.
  • the three-dimensional muscle tissue of the present invention does not have to contain heme. This is because the present invention does not require a heme to supply oxygen to the cells. It should be noted that heme that is slightly mixed during cell collection and heme that is added not for the purpose of supplying oxygen but for coloring and flavoring are treated as additives and are not regarded as heme in the present invention.
  • Example 1 Preparation of Support Member with Anchor
  • the support member 8 or 9 and the muscle tissue anchor 11 are vertically arranged in a mesh shape of three rows, and the support member with an anchor is arranged.
  • the muscle tissue anchor 11 fixes the contracting muscle tissue
  • the support members 8 and 9 supply nutrients to the inside of the muscle tissue by the culture solution.
  • the first connector 4 and the second connector 5 fix the muscle tissue anchor 11 and the first support member 8 or the second support member 9.
  • the hole through which the flow path forming member 10 is passed is closed with a sealing material 20 (Ecoflex (registered trademark) manufactured by BASF), but the second connector 5 is used. Since there is a hole through which the channel forming member 10 passes, the culture solution that has passed through the channel is mixed with the culture solution in the culture space and discharged from the culture solution discharge port.
  • a sealing material 20 Ecoflex (registered trademark) manufactured by BASF
  • the support member with an anchor shown in FIG. 4 was manufactured using a stereolithography machine (DigitalWax028J, DWS).
  • the resin used for DWS was a DWS exclusive resin (DM210).
  • the muscle tissue anchor 11, the first support member 8 and the second support member 9 were coated with PMBV631 (2 wt% ethanol) and then coated overnight with fibronectin (10 ⁇ L), which promotes cell adhesion. Acupuncture needles were coated with bovine serum albumin (BSA, 1%) for 1 hour to suppress cell adhesion.
  • BSA bovine serum albumin
  • C2C12 myoblast was used. 2.0 ⁇ 10 6 cells were seeded on a 150 mm dish, and the cells passaged 2 days later were used as cells, all of which were P11 (11 passages) or less. The cell concentration was 4.0 ⁇ 10 7 cells / mL.
  • Injection of hydrogels containing skeletal myoblasts (C2C12) into the device was performed as follows. After adding 3 mL of trypsin to the dish in which C2C12 was growing, the cells were detached from the dish by incubating at 37 ° C for 5 minutes. After adding 7 mL of Dulbecco's modified Eagle's medium (DMEM) to the dish, pipette twice with an electric pipette to collect 50 mL. The cells were precipitated by centrifugation at 200 g for 3 minutes. Subsequent work was done on ice.
  • DMEM Dulbecco's modified Eagle's medium
  • parallel culture means culturing with the flow path 3 perpendicular to the direction of gravity.
  • the two anchored support members are arranged facing each other, and the first connector 4 and the second connector 5 are fixed to the base material 17.
  • An acupuncture needle (SJ-217, manufactured by Seirin Corporation) is inserted into the first support member and the second support member, and the side wall 18 is attached to the base material 17 to form the culture space 2.
  • 3. 3. Inject hydrogel containing C2C12 into the culture space and incubate for 30 minutes at 37 ° C to solidify the hydrogel.
  • 4. 3. 3. Place the device obtained in step 1 in a 25 mL tube, soak it in the culture medium, and let it stand for 30 minutes. 5.
  • the acupuncture needle is pulled out to form a channel 3. 6. 5. Transfer the device obtained in step 1 to a dish and remove the side wall 18. 7. The sealing material 20 closes the hole for inserting the needle on the culture solution introduction port 6 side. 8.
  • One perfusion tube is connected to the culture solution inlet 6, and the other perfusion tube is placed near the liquid surface to perfuse the culture solution.
  • the two perfusion tubes are connected to a syringe pump, and the culture solution is perfused by the syringe pump. In the above "8.”, the other perfusion tube is inserted below the culture solution surface, but it may be connected to the culture solution discharge port 7.
  • vertical culture means culturing with the flow path 3 parallel to the direction of gravity.
  • the two anchored support members are arranged facing each other, and the first connector 4 and the second connector 5 are fixed to the base material 17.
  • An acupuncture needle (SJ-217, manufactured by Seirin Corporation) is inserted into the first support member and the second support member, and the side wall 18 is attached to the base material 17 to form the culture space 2.
  • 3. 3. Inject hydrogel containing C2C12 into the culture space and incubate for 30 minutes at 37 ° C to solidify the hydrogel. 4.
  • the perfusion tube is connected to the culture solution inlet 6. 5. 4. Place the device obtained in step 1 in a 100 mL tube and bring the electrodes closer together.
  • the tube was shaken with a shaker and rotated at 60 rpm for culture.
  • the oxygen concentration that can be taken in only from the surface of the culture solution can be made constant in the culture solution.
  • two syringe pumps to which the tubes are connected are prepared, the tube for injecting the medium is placed above the liquid surface, and the tube for sucking the medium is brought close to the bottom, for suction and injection.
  • tissue sections of three-dimensional muscle tissue were prepared. Section preparation was performed in the order of (i) cryoprotect treatment, (ii) freezing treatment, (iii) section preparation, (iv) HE staining, and (v) immunostaining.
  • Cryoprotect treatment is a treatment to prevent the formation of ice particles in the tissue when frozen.
  • PBS refers to phosphate buffered saline.
  • Freezing treatment Frozen treatment is performed before preparation of frozen sections. 1. Pour liquid nitrogen into a heat insulating container (height 4-5 cm). 2. Place the cryodish in liquid nitrogen using tweezers. 3. If you soak in liquid nitrogen for a long time, the 3D muscle tissue will crack, so take the cryodish in and out every 1-2 seconds to gradually harden the 3D muscle tissue.
  • Section preparation Using a cryostat section is prepared to a thickness of 8 ⁇ m.
  • 1. Place the cryodish containing the frozen 3D muscle tissue sample in the cryostat and warm to -20 ° C. 2. Remove the sample from the cryodish and make a short side with a thickness of 8 ⁇ m. 3. Rotate the sample 180 degrees to make a short side with a thickness of 8 ⁇ m on the opposite side. 4. Rotate the sample 90 degrees to make 3 to 4 long sides with a thickness of 8 ⁇ m. 5. Cut the sample by 200 ⁇ m and repeat the preparation of the long side until the long side disappears. 6. Attach the obtained section to MAS coated glass.
  • HE staining The procedure for HF staining is as follows. 1. Dry for 1 day after making the section. 2. Soak the section in Meyer's hematoxylin solution and let stand for 5 minutes. 3. Adsorb and remove excess hematoxylin solution on paper. 4. Transfer the sections to a stain bottle containing water and remove excess stain. 5. Soak in warm water at 50 ° C and let stand for 5 minutes. 6. Transfer the sections to a stain bottle containing water. 7. Soak in eosin solution and let stand for 5 minutes. 8. Soak in 100% ethanol and slowly move the glass slide up and down 5 times to remove excess stain. 9. Repeat step 8 above, changing the bottle, a total of 3 times. 10. Soak in xylene (3 times for 5 minutes each). 11. Enclose a few drops of canard new in a section and attach a cover glass. 12. Allow to dry overnight.
  • the three-dimensional muscle tissue prepared by the present invention is characterized by being oriented.
  • the orientation index evaluation of the three-dimensional muscle tissue was performed by the following procedure using the two-dimensional Fourier transform. 1. Cut so that the vertical and horizontal dimensions are power pixels of 2. 2. Make it gray. 3. Put on the honey window. 4. Two-dimensional Fourier transform is performed using the function fft2 of numerical analysis software (MATLAB (registered trademark), provided by MathWorks). 5. Integrate the 2D Fourier transform image for polar constellation and add the pixel values from 1 degree to 180 degrees. 6. From 1 degree to 180 degrees, divide by the sum of the totals to get the average.
  • MATLAB registered trademark
  • nuclei were discriminated from the HE-stained image using image processing software (ImageJ, open source), and the number of nuclei was counted according to the following procedure. 1. Combine the images using Make composite. 2. Convert to 32-bit RGB color using RGB Color. 3. Use Deconvolution to split the RGB color image for HE. 4. Use Threshold for the Blue image to separate the image containing the nucleus from the background. 5. Separate the connected nuclei using Watershed. 6. Count the number of nuclei.
  • FIG. 10 shows the results of flowing ink through the flow path 3 in vertical culture and parallel culture.
  • FIG. 13 is a photograph in which the tissue after 36 hours is embedded in an alginate gel and an enlarged photograph of a portion of the mesh anchor 11. It was shown that the muscle tissue was anchored even when tension was applied so that the width contracted by 39%.
  • FIG. 14 (a) a fluorescence-stained image of the part of the muscle tissue in contact with the medium.
  • Red is the actin filament dyed with phalloidin
  • blue is the nucleus dyed with Hoechst. It can be confirmed that many nuclei are on one myotube, and it can be seen that myoblasts are fused.
  • FIG. 14 (b) is a diagram showing the directional distribution of actin from the image of only the actin filament. Since it can be confirmed that the peak stands at 0 degrees, it can be seen that the muscle tissue is oriented in the direction of O degrees.
  • FIG. 15 shows the results of cutting the muscle tissue after culturing and observing the live / dead assay with a confocal microscope. Since it is possible that cells die on the cut surface, the inside of the tissue was observed from the 34 ⁇ m cut surface. There were 196 live cells and 725 dead cells, and the survival rate was as low as 21%. The cause of the low cell viability may be damage during tissue cutting. We are observing the tissue inside 34 ⁇ m from the cut surface of the tissue, but it is possible that the tissue is pulled during cutting and damages the cells.
  • Another characteristic result is that there are many live cells between the channels, but there are many dead cells around the channels.
  • the oxygen deficiency in culture which is considered to be the cause of cell death
  • cells near the channel should be alive, and the number of dead cells should increase as the cells move away from the channel, but the opposite tendency was shown. Therefore, it is considered that the cells did not die during the culture period, but the cells around the channel were damaged and died when the acupuncture needle was pulled out to form the channel. Since the needle is BSA coated, the cells should not be adhered, but it is possible that the cells died due to rubbing when the needle was pulled out. To avoid this, it is necessary to slowly pull out the needle.
  • FIG. 16 is a graph of the change in width during culture without shaking. Those that were perfused with an anchor, those that were not perfused with an anchor, and those that were not perfused without an anchor were with perfusion (hereinafter, w / perfusion), without perfusion (hereinafter, w / o perfusion), and without anchor, respectively. (Hereafter, w / o anchor).
  • the width was observed by looking at the side of the tube, that is, the curved surface with a camera (VW-600C, KEYENCE). The length was calculated based on the referenceable length of the anchored support member array. The experimental results are the following two.
  • the anchor 11 prevents the muscle tissue from falling off in the device of the present invention for culturing the contractile muscle tissue.
  • FIG. 18 shows a plot with time taken on the horizontal axis. There are four channels in the tissue, but since they appear to overlap, only two should actually be visible.
  • FIG. 21 (a) is an HE-stained image. Since it was a sample that confirmed that perfusion had failed in the ink flow experiment, it was confirmed that there was no nucleus in the center. It is considered that the non-existence is either that the nucleus is enucleated and no longer exists, or that the culture solution is sought and moved to the peripheral part. In particular, as shown in FIG.
  • the initial cell concentration of the sample used in this experiment was 4.0 ⁇ 10 7 cells / mL, and considering that the cross section of 16 mm 2 shrank to about 1.3 mm 2 , the cell concentration was 49 ⁇ 10 7 cells / mL. Is.
  • FIG. 23 (a) is a sample with perfusion
  • FIG. 23 (b) is a sample without perfusion.
  • FIG. 23 (a) it can be seen that there is a hole.
  • the cell concentrations in the rectangular portion of 100 ⁇ m ⁇ 50 ⁇ m were compared for both samples (Fig. 23 (c)).
  • Fig. 23 (c) it was found that the cell concentration decreased in both samples as they moved away from the edge of the tissue, but the decrease in cell concentration slowed in the sample with perfusion. This is thought to be due to the fact that oxygen and nutrients were also supplied from the inside by perfusion.
  • FIG. 25 shows the actin filaments and nuclei on the surface of the tissue, and the lower part shows the inside of the tissue similarly dyed.
  • the results of the sample, w / o anchor sample are shown.
  • FIG. 26 shows the orientation index of the lower actin filament. It was found that the sample with anchor and perfusion (with perfusion) had muscle tissue with a high orientation index.

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Abstract

En fournissant un canal pour transporter une solution de culture à l'intérieur d'un hydrogel contenant des myoblastes squelettiques, les présents inventeurs ont réussi à cultiver un tissu musculaire tridimensionnel ayant une épaisseur considérable et étant formé de fibres musculaires orientées dans la même direction. Le tissu musculaire tridimensionnel produit conformément à la présente invention devrait avoir une texture similaire à celle de la viande pour les steaks. Dans l'application à la médecine régénérative, on s'attend à ce qu'un dommage important et épais du tissu musculaire puisse être réparé par un seul traitement grâce à l'utilisation du tissu musculaire tridimensionnel ayant une épaisseur considérable.
PCT/JP2021/032244 2020-09-15 2021-09-02 Dispositif de formation de tissu musculaire tridimensionnel et procédé de production de tissu musculaire tridimensionnel WO2022059498A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010539935A (ja) * 2007-09-24 2010-12-24 ノーティス，インク． 灌流可能な微小血管システムを作製するための方法
JP2019122335A (ja) * 2018-01-18 2019-07-25 国立大学法人 東京大学 人工三次元組織のバリア機能測定システム、人工三次元組織のバリア機能測定方法及び人工三次元組織を用いた薬剤評価方法
WO2020179257A1 (fr) * 2019-03-04 2020-09-10 日清食品ホールディングス株式会社 Tissu musculaire tridimensionnel et son procédé de production

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010539935A (ja) * 2007-09-24 2010-12-24 ノーティス，インク． 灌流可能な微小血管システムを作製するための方法
JP2019122335A (ja) * 2018-01-18 2019-07-25 国立大学法人 東京大学 人工三次元組織のバリア機能測定システム、人工三次元組織のバリア機能測定方法及び人工三次元組織を用いた薬剤評価方法
WO2020179257A1 (fr) * 2019-03-04 2020-09-10 日清食品ホールディングス株式会社 Tissu musculaire tridimensionnel et son procédé de production

Non-Patent Citations (1)

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Title
ISHII, Y. ET AL.: "FORMATION OF MICRO-SEE PERFUSABLE CHANNELS IN MM- THICK MUSCLE TISSUE", MEMS 2020, VANCOUVER, CANADA ., 18 January 2020 (2020-01-18), pages 456 - 458, XP033753102, DOI: 10.1109/MEMS46641.2020.9056142 *

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